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The Journal of Immunology, 2005, 175: 3584-3593.
Copyright © 2005 by The American Association of Immunologists

Interplay of Cytokines and Microbial Signals in Regulation of CD1d Expression and NKT Cell Activation1

Markus Sköld*, Xiaowei Xiong*, Petr A. Illarionov{dagger}, Gurdyal S. Besra{dagger} and Samuel M. Behar2,*

* Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and {dagger} School of Biosciences, University of Birmingham, Edgbaston, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study we show that like MHC class I and class II molecules, cell surface CD1d expression on APC is regulated and affects T cell activation under physiological conditions. Although IFN-{gamma} alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-{gamma} and a second mediated by microbial products or by the proinflammatory cytokine TNF. IFN-{gamma}-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. APC expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independently of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. Our findings support a model in which CD1d induction regulates NKT cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antigen-presenting molecules play a fundamental role in T cell development, T cell priming, and regulation of immunity. The cell surface expression of Ag-presenting molecules is particularly critical for host defense against intracellular pathogens as the presentation of microbial Ags by class I or class II MHC molecules is the principle way that the immune system identifies infected cells. Although Ag-presenting molecules are constitutively expressed by many cells, they are also regulated by various stimuli such as cytokines and microbial products.

MHC class II is constitutively expressed by B cells, dendritic cells (DC),3 and macrophages (M{phi}); it is also induced by cytokines, principally IFN-{gamma}, on a variety of cells including M{phi}, endothelial, and epithelial cells (1). MHC class II is also modulated during cell differentiation as exemplified by its redistribution from the intracellular MHC class II compartment to the cell surface during DC maturation (2). The class II MHC redistribution that accompanies DC maturation can be induced by TNF or by microbial products such as the TLR4 ligand LPS (3, 4, 5).

Although MHC class I is constitutively expressed by nearly every nucleated cell, it too is regulated by cytokines. IFN-{gamma} increases cell surface levels of MHC class I on a variety of cell types and regulates transcription of many proteins involved in the MHC class I Ag-processing pathway (1, 6). These effects of IFN-{gamma} are critical in T cell responses to viral pathogens as the infected cell makes itself a suitable target of CD8+ T cells by increasing the efficiency of Ag processing and the number of Ag-MHC complexes expressed at the cell surface. The importance of class I MHC-antigenic peptide complexes for the host is best appreciated by the number of human viruses for which their success as pathogens is related to their interference with class I MHC expression and Ag processing (7).

Like class I and class II MHC, the Ag-presenting molecule CD1d is constitutively expressed by many cell types. It is prominently expressed by splenic B cells, and CD1d is found on M{phi}, DC, and even T cells (8). Whether cell surface expression of CD1d is subject to additional regulation by cytokines or microbial products, as observed for MHC, is an important question. Although class I and class II MHC induction increases the efficiency of T cell activation, it is unknown whether increased surface expression of CD1d will do the same. The observation that mice lacking CD1d-restricted NKT cells have diminished host resistance to certain pathogens, impaired tumor immunity, and alterations in their predisposition to autoimmune disease indicates that NKT cells participate in these immunological responses (9, 10). There are two emerging models of how lipid Ag presentation by CD1d activates NKT cells.

NKT cell recognition of foreign lipids has been modeled using the synthetic Ag {alpha}-galactosylceramide ({alpha}-GalCer), which binds to CD1d and specifically activates invariant NKT (iNKT) cells that are defined by their canonical V{alpha}14-J{alpha}18 TCR-{alpha} chain (murine) or V{alpha}24-J{alpha}18 TCR-{alpha} chain (human) (11, 12). In theory, microbial glycolipid Ags are so sufficiently different from host lipids that CD1d-restricted NKT cells can recognize them as foreign. Recently, several microbial lipid Ags have been identified that are presented by CD1d and activate NKT cells including monoglycosylceramides from Sphingomonas species, phosphatidylinositol mannosides from Mycobacterium tuberculosis, and lipophosphoglycan from Leishmania donovani (13, 14, 15, 16).

In addition to recognizing microbial lipids, both iNKT and NKT cells expressing diverse TCR recognize CD1d in the absence of exogenously added Ags (17). CD1d autoreactivity is Ag-dependent and the lysosomal glycosphingolipid isoglobotrihexosylceramide (iGb3) has recently been suggested to be the primary self-Ag for CD1d-autoreactive human and murine iNKT cells (18, 19). Cellular phospholipids also bind to CD1d, and reactivity to phosphatidylethanolamine, phosphatidylinositol, and related Ags does occur for some iNKT and NKT cells (18, 20). NKT cell recognition of self-lipids may be particularly important during noninfectious immunological responses including tumor immunity and autoimmunity. Recognition of self-lipid Ags may even be important for NKT cell activation during infection because IL-12 induced by microbial pathogens costimulates activation of CD1d autoreactive human iNKT cells in vitro (21). Heat killed Salmonella presented by DC lacking the iGb3 endogenous Ag or an intact TLR signaling pathway does not activate murine iNKT cells (16). These data support the paradigm that one pathway for iNKT cell activation is the costimulation of weak self-Ag recognition by pathogen-induced IL-12 production. Importantly, recognition of self-lipid Ags also requires that NKT cell activation must be regulated to avoid inappropriate responses.

In studies designed to better understand how NKT cells are activated by infection, we discovered an IFN-{gamma}-dependent mechanism by which bacterial infection or exposure to microbial products, including TLR ligands, induces CD1d expression on M{phi} in vitro and in vivo. The effect of TLR ligands and live bacteria on CD1d induction is mediated by the proinflammatory cytokine TNF. This observation suggested that modulation of cell surface CD1d levels on APC could be a mechanism that regulates NKT cell activation. We found that both NKT cell hybridomas and primary NKT cells are activated more efficiently by APC expressing higher cell surface CD1d levels. Both iNKT cell and diverse NKT cell populations share this mechanism and it is independent of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. These data provides a new basis for understanding how CD1d-restricted NKT cells are activated during inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

B6 and B6.129S7-Ifngrtm1Agt/J (IFN-{gamma}R–/–) mice were obtained from The Jackson Laboratory. Mice were housed under specific pathogen-free conditions and were used in a protocol approved by the institution. Six- to 8-wk-old B6 mice were used as a source of primary NKT cells and as recipient mice in the M{phi} adoptive transfer experiments described below. Mycobacterium tuberculosis (Mtb)-infected mice were housed in a biosafety level 3 facility at the Animal Biohazard Containment Suite (Dana-Farber Cancer Institute) and were used in an approved protocol. B6 and IFN-{gamma}R–/– mice were used as a source of inflammatory M{phi}.

M{phi} and in vitro cultures

Inflammatory M{phi} were elicited by i.p. injection of 1-ml sterile 3% thioglycolate medium (REMEL). Peritoneal exudate cells were harvested by lavage after 4 days. M{phi} were purified from peritoneal exudate cells by magnetic cell sorting with CD11b microbeads (Miltenyi Biotec). Positively selected cells routinely contained ~95% F4/80+CD11b+ M{phi} as determined by flow cytometry. Enriched M{phi} (1 x 106) were seeded into 24-well plates in complete culture medium (RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS (HyClone), penicillin/streptomycin, L-glutamine, sodium-pyruvate, 2-ME, nonessential amino acids, essential amino acids, and HEPES buffer, all from Invitrogen Life Technologies). Synthetic TLR2 ligand Pam3Cys-Ser-(Lys)3 x 3 HCl was obtained from EMC and ultra pure TLR4 ligand Escherichia coli LPS (O111:B4 strain) from InvivoGen. Recombinant mouse IFN-{gamma}, IL-12p70, and TNF (US Biological) were used at the concentrations indicated. Supernatants and M{phi} were harvested at the time points indicated after cell culture at 37°C, 5% CO2. M{phi} were incubated with 2 mM EDTA in PBS for 10 min at 37°C to detach adherent cells for flow cytometry. NaN3 (2 mM) was added to the supernatant before their cytokine content was measured using ELISA (reagents from eBioscience). To examine the contribution of TNF in CD1d regulation by TLR agonists and live Mtb, a blocking anti-TNF mAb (MP6-XT3) and an isotype control Ab (no azide/low endotoxin; BD Pharmingen) were used (40 µg/ml).

Bacteria and infections

An in vitro infection model was used to analyze the effect of live Mtb on CD1d and class II MHC expression by inflammatory M{phi}. Virulent Mtb (H37Rv) or avirulent Mtb (H37Ra strain) was grown to mid-log phase in Middlebrook 7H9 medium containing 10% albumin/dextrose/catalase enrichment (both from BD Biosciences). Bacteria were opsonized using RPMI 1640 medium with 2% human serum (Gemini Bio-Products), 10% FBS, and 0.05% Tween 80 and then washed twice with complete medium without antibiotics. Bacteria were passed through a 5-µm syringe filter (Millipore), counted in a Petroff-Hausser chamber and added to purified M{phi} at different multiplicity of infection (MOI) as indicated. M{phi} and bacteria were cultured in complete medium without antibiotics in the presence or absence of rIFN-{gamma} (2.5 U/ml). In some experiments, bacteria were heat killed at 80°C for 20 min. The M{phi} phenotype was analyzed by flow cytometry, and cytokine production was analyzed by ELISA after 24 or 72 h incubation. Mice were infected with virulent Mtb (Erdman strain) via the aerosol route as previously described (22).

T cell hybridoma and primary NKT cell assays

All CD1d-restricted T cell hybridomas used have been previously described (18, 23, 24). Hybridomas KT/7, KT/12, KT/22, and KT/23 were kindly provided by Dr. S. Cardell (Lund University, Lund, Sweden) and hybridoma DN32.D3 by Dr. A. Beandelac (University of Chicago, Chicago, IL). M{phi} were plated in flat-bottom 96-well plates and kept in complete medium alone, or with rIFN-{gamma} (2.5 U/ml), Pam3Cys (1 ng/ml), or both for 3 days before the T cell hybridoma cells were added. CD1d autoreactivity was tested by seeding M{phi} at different cell densities. A constant number of M{phi} were used to test for {alpha}-GalCer reactivity. Gal({alpha}1->2)GalCer ({alpha}-GalGalCer) was added to M{phi} the last 6 h of the 3-day culture period. The complete synthesis of {alpha}-GalGalCer will be reported elsewhere (P. A. Illarionov and G. S. Besra, unpublished observations). A complete structural analysis of {alpha}-GalGalCer was performed by nuclear magnetic resonance and electrospray mass spectrometry (data not shown). Cells were washed once before the T cell hybridomas (5 x 104/well) were added. RMA-S.CD1d cells were sorted into different sublines based on their CD1d surface expression. These RMA-S.CD1d sublines or RMA-S nontransfected cells were seeded into 96-well plates (105/well). In some assays, RMA-S cells were pulsed with 10 ng/ml {alpha}-GalCer for 6 h at 37°C before adding {alpha}-GalCer-reactive T cell hybridomas (105/well). T cell hybridoma assays were incubated for 24 h at 37°C, 5% CO2, before IL-2 was measured in the culture supernatants by ELISA (reagents from BD Pharmingen). CD1d cell surface expression by M{phi} and RMA-S cells was monitored in each experiment using flow cytometry.

Primary NKT cells were enriched by depleting thymocytes of CD8+ cells using magnetic cell sorting with CD8{alpha}-microbeads (Miltenyi Biotec). Enriched thymic NKT cells (0.2–1 x 106) were cultured with {alpha}-GalCer/vehicle-pulsed APC (105/well), with or without rIL-2 (Chiron) at 100 U/ml as indicated. RMA-S cells were irradiated at 2000 rad before they were used as APC. IL-4 and IFN-{gamma} was measured in the culture supernatants by ELISA (reagents from BD Pharmingen) after incubation for 48 h at 37°C, 5% CO2.

Detection of CD1d regulation in vivo

An adoptive transfer system was used to examine CD1d and class II MHC regulation at the site of infection in vivo. Purified thioglycolate elicited peritoneal M{phi} were obtained from B6 or IFN-{gamma}R–/– mice as described earlier and labeled with 1 µM CFSE (Molecular Probes). A total of 1 x 107 CFSE-labeled M{phi} were injected i.v. via the tail vein into Mtb-infected B6 recipients or age- and sex-matched uninfected B6 mice. Transfers were performed 4–5 wk after aerosol infection during the peak of the immune response. Three or five recipient mice were used per group in five separate experiments. At 48 or 72 h after cell transfer, single cell suspensions were prepared from lung tissue as described with the modification that collagenase digested lung tissue was treated with 200 U/ml DNase I (Sigma-Aldrich) for 10 min at 37°C (22).

Detection of iNKT cell activation in vivo

An adoptive transfer system using {alpha}-GalCer-pulsed M{phi} and an IFN-{gamma} ELISPOT assay was adapted from Fujii et al. (25) and used to detect activation of iNKT cells in vivo. Purified thioglycolate-elicited peritoneal M{phi} were obtained from B6 mice as described above and cultured in medium alone or with rIFN-{gamma} (2.5 U/ml) and Pam3Cys (1 ng/ml) for 3 days. M{phi} were pulsed with {alpha}-GalCer (100 ng/ml) for 6 h, washed with PBS and injected i.v. via the tail vein into naive B6 recipients (106 M{phi}/mouse). At 48 h after M{phi} adoptive transfer, single cell suspensions were prepared from the spleens of the recipient mice or from naive B6 control mice. Total splenocytes were cultured with {alpha}-GalCer (100 ng/ml) or with vehicle (DMSO) in ELISPOT plates precoated with anti-IFN-{gamma} capture mAb (all ELISPOT reagents from BD Pharmingen). The cells were incubated for 16 h at 37°C, 5% CO2, before they were discarded and the plates washed with deionized water and PBS/Tween 20. The plates were incubated with a biotinylated secondary mAb, washed, and incubated with streptavidin-HRP. After several washes the plates were developed using 3-amino-9-ethylcarbazole substrate. The spots were enumerated using an ImmunoSpot plate reader and ImmunoSpot software version 3 (Cellular Technology).

Flow cytometry

PBS with 1% w/v BSA and 2 mM NaN3 were used to wash the single-cell suspensions and to dilute Abs and second step reagents. Cells were incubated with purified anti-CD16/CD32 (2.4G2; ATCC HB-197) at 25 µg/ml to inhibit nonspecific staining. The following PE-, FITC-, or PE-Cy5-conjugated, or biotinylated mAbs and second step reagents were obtained from BD Pharmingen: anti-CD1d (1B1), anti-I-A/I-E (M5/114.15.2), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD22.2 (Cy34.1), anti-CD40 (3/23), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-Ly6C/G (RB6-8C5), and appropriate isotype control Abs and streptavidin. PE-conjugated anti-CD115 (AFS98) was purchased from eBioscience and FITC-conjugated anti-CD205 (MCA949F) was purchased from Serotec. The F4/80 mAb (ATCC HB-198) and the M1/42 mAb (ATCC TIB-126) were purified and biotinylated using standard protocols or were conjugated to Alexa Fluor 488 using a labeling kit from Molecular Probes. Stained cells were washed and analyzed directly or fixed overnight at 4°C in 1% paraformaldehyde in PBS. Cells were collected using a FACSort or a FACSCanto flow cytometer (BD Biosciences) and analyzed using CellQuest (BD Biosciences) or FlowJo software (Tree Star).

Real-time PCR

M{phi} were purified and cultured as described above. After 24 h of culture, total RNA was prepared using TRIzol Reagent (Invitrogen Life Technologies) followed by DNase I (amplification grade) treatment according to the manufacturer’s instructions (Invitrogen Life Technologies). RNA was reverse transcribed to single-stranded cDNA using SuperScript II reverse transcriptase and oligo(dT) to prime first-strand synthesis (Invitrogen Life Technologies). The following primers were used for real-time PCR amplification using an iTaq SYBR Green Supermix kit from Bio-Rad and an iCycler real-time PCR detection system (Bio-Rad): CD1d forward 5'-TGTCACCTAAAGAAGACTATCCCATTG-3', reverse 5'-CCGAAGCATTCCCAGGGTA-3'; and {beta}-actin forward 5'-CATCTTGGTTAGACTTGCCCAT-3', reverse 5'-GGAGACCACGGACAAATAGGG-3'. Samples were incubated at 50°C for 2 min followed by 10 min incubation at 95°C. Each amplification cycle consisted of 15 s at 95°C and 1 min at 60°C and was repeated 40 cycles. The relative amount of CD1d mRNA was determined by the threshold cycle {Delta}{Delta}Ct method relative to the expression of the housekeeping gene {beta}-actin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cytokine mediated up-regulation of CD1d on elicited peritoneal M{phi}

Several pathogens cause worse disease in mice that lack CD1d or iNKT cells, suggesting that CD1d-restricted NKT cells play a physiologic role in host defense (9). Implicitly, these studies indicate that following infection CD1d-restricted NKT cells must become activated. Modulation of CD1d cell surface expression on APC may be one mechanism that regulates NKT cell activity. Therefore, we developed an in vitro model using elicited peritoneal M{phi} to test whether bacterial infection and inflammatory mediators modulate cell surface expression of CD1d.

Highly purified F4/80+CD11b+ M{phi} expressed low levels of CD1d and were heterogeneous for class II MHC (data not shown). These M{phi} expressed CD115 and low levels of CD40, CD80, and CD86, but not Ly6C/G, CD11c, or CD205 (data not shown). M{phi} were infected with Mtb, and CD1d and class II MHC expression was analyzed 72 h later. Infection of highly purified M{phi} did not affect cell surface expression of CD1d or MHC class II (Fig. 1A).



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  		FIGURE 1. CD1d up-regulation on Mtb-infected M{phi} is IFN-{gamma}-dependent. A, Thioglycolate-elicited purified peritoneal M{phi} were infected in vitro with live Mtb (H37Ra) at the indicated MOI in the presence (circles) or absence (squares) of rIFN-{gamma} (2.5 U/ml). CD1d (closed symbols, left) and class II MHC (closed symbols, right) expression on F4/80+ M{phi} was analyzed on day 3. Values are the MFI. B, Representative dot plots show the expression of CD1d (top panels) and class II MHC (bottom panels) by F4/80+ purified M{phi} cultured for 3 days in vitro alone, or in the presence of rIFN-{gamma}, live Mtb (MOI 5:1), or both rIFN-{gamma} and bacteria. C, CD1d (closed symbols, left) or class II MHC (closed symbols, right) expression by purified F4/80+ inflammatory M{phi} was analyzed 4 days after culture with rIFN-{gamma} (circles), rIL-12-p70 (triangles), or rTNF (squares). Open symbols show Ab isotype control staining.

 
IFN-{gamma} is the principal M{phi} activating factor and affects multiple aspects of M{phi} function including induction of bactericidal activity and cell surface expression of class II MHC, which facilitates recognition by CD4+ T cells. Microarray analysis of bone marrow-derived M{phi} has previously shown that transcription of CD1d and MHC class II genes are induced following stimulation with IFN-{gamma} and Mtb (see supplemental data in Ref.26). Therefore, we tested the possibility that IFN-{gamma} treatment of Mtb-infected M{phi} would induce cell surface expression of CD1d and class II MHC. When M{phi} were infected with Mtb in the presence of low amounts of rIFN-{gamma}, CD1d was induced in a dose-dependent manner (Fig. 1A). Uninfected M{phi} cultured in the absence of rIFN-{gamma} expressed low CD1d levels as mean fluorescence intensity (MFI = 22), whereas heavily infected M{phi} cultured in the presence of rIFN-{gamma} expressed high CD1d levels (MFI = 238), representing more than a 10-fold increase in CD1d cell surface expression. Under these conditions, the induction of class II MHC by rIFN-{gamma} was unaffected by the bacteria, except at the highest MOI where Mtb had a slight inhibitory effect on IFN-{gamma}-induced class II MHC surface expression (Fig. 1, A and B). Still, the inhibition of class II MHC was modest compared with induction of CD1d under the same culture conditions. Similar results were obtained using the virulent H37Rv strain of Mtb (data not shown). These data demonstrate that CD1d expression by M{phi} is not static and that microbial pathogens and IFN-{gamma} act synergistically to induce cell surface CD1d.

Even in the absence of bacteria, rIFN-{gamma} induced CD1d and class II MHC on uninfected M{phi} (Fig. 1, A and B). To determine theoptimal conditions for the maximal induction of CD1d and class II MHC by rIFN-{gamma} on cultured inflammatory M{phi}, rIFN-{gamma} was titrated over a large concentration range. rTNF and rIL-12p70 were also individually tested as these cytokines can be produced by infected M{phi} and might also modulate CD1d and class II MHC expression. Even at low concentrations, rIFN-{gamma} was sufficient to up-regulate CD1d on M{phi} and did so in a dose-dependent manner (Fig. 1C). In contrast, rTNF and rIL-12p70 had no effect on CD1d expression. Treatment with rIFN-{gamma} led to a 4-fold induction of CD1d compared with medium treated M{phi}. MHC class II was also up-regulated by rIFN-{gamma} as expected, and to some extent by rIL-12p70 at the highest concentration (Fig. 1C).

The comparison between CD1d and class II MHC expression identifies a difference in the regulation of these two Ag-presenting molecules. Maximum cell surface expression of class II MHC was elicited by rIFN-{gamma} alone, whereas optimal induction of CD1d requires two signals mediated by rIFN-{gamma} and a microbial signal.

Synergistic effect by IFN-{gamma} and TLR agonists on CD1d cell surface expression

What bacterial factor(s) regulate CD1d? The observed induction of CD1d did not require actively metabolizing bacteria and the inducing factor was heat stable because heat killed Mtb also induced CD1d in an IFN-{gamma}-dependent manner (Fig. 2A). As observed with viable bacteria, CD1d was not significantly induced in the absence of rIFN-{gamma}, even at a ratio of 50:1 (heat killed bacteria to M{phi}) (data not shown). A reverse correlation between CD1d and class II MHC expression was observed following treatment of M{phi} with heat killed bacteria and rIFN-{gamma}. Although this combination dramatically induced CD1d expression, class II MHC was inhibited in a dose-dependent manner (Fig. 2A). Microbial pathogens including Mtb are detected by the immune system via TLR signaling. Although TLR agonists alone did not affect CD1d expression, low concentrations of Pam3Cys (a TLR2 agonist) or LPS (a TLR4 agonist) both dramatically induced CD1d in the presence of rIFN-{gamma} (Fig. 2B). Similar to heat killed bacteria, TLR agonists inhibited IFN-{gamma}-induced class II MHC up-regulation as has been previously reported (27). In the absence of IFN-{gamma}, LPS but not Pam3Cys, induced class I MHC expression in a dose-dependent manner (Fig. 2B). IFN-{gamma} alone also induced class I MHC, and addition of either TLR agonist had a minor effect on class I MHC cell surface expression. Under these conditions, CD1d induction could be detected within 24 h and was maximal within 2–3 days (Fig. 2C). Regulation of CD1d expression appears to be at the RNA level because Pam3Cys alone had little effect on expression. In contrast, treatment with IFN-{gamma} led to an increase in CD1d RNA, and IFN-{gamma} plus Pam3Cys synergistically increased the amount of CD1d RNA relative to {beta}-actin (Fig. 2D).



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  		FIGURE 2. IFN-{gamma} and bacterial products have a synergistic effect on CD1d induction. CD11b+ inflammatory M{phi} were cultured alone or with heat killed (HK) Mtb (H37Ra strain) at the indicated MOI (A); or with the TLR2 agonist Pam3Cys or the TLR4 agonist LPS at the indicated concentrations (B) in the presence (circles) or absence (squares) of rIFN-{gamma} (2.5 U/ml). CD1d (closed symbols, left), class II MHC (closed symbols, center), and class I MHC (closed symbols, right) expression by F4/80+CD11b+ M{phi} was examined on day 3. The mean MFI ± SEM of three to four experiments is shown in B. C, Purified M{phi} were cultured alone (squares) or stimulated with Pam3Cys (1 ng/ml) and rIFN-{gamma} (2.5 U/ml) (circles) for 24, 48, or 72 h. CD1d expression (filled symbols) by F4/80+ M{phi} was determined by flow cytometry. Open symbols show Ab isotype control staining. D, Alteration of CD1d RNA levels relative to {beta}-actin were determined by real-time RT-PCR. Fold increase was calculated by the {Delta}{Delta}Ct method.

 
Synergistic effect of TNF and IFN-{gamma} in CD1d regulation

In addition to its synergistic effect on CD1d and class II MHC expression, the combination of rIFN-{gamma} and heat killed bacteria or TLR agonists also modulates cytokine production by M{phi} (Fig. 3A and data not shown). Highly purified M{phi} cultured with TLR agonists did not secrete detectable amounts of IL-12p40 and TNF, and only small amounts of IL-6. Interestingly, the induction of all three cytokines by TLR agonists was IFN-{gamma}-dependent.



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  		FIGURE 3. TNF and IFN-{gamma} synergize to up-regulate CD1d on inflammatory M{phi}. A, rIFN-{gamma} modulates cytokine production by inflammatory M{phi} in response to TLR ligands. M{phi} were stimulated with the TLR2 agonist Pam3Cys in the absence ({circ}) or presence (•) of rIFN-{gamma} (2.5 U/ml). IL-6, TNF, and IL-12p40 were measured in the culture supernatants on day 3. B, Purified M{phi} were cultured with rTNF in the presence (circles) or absence (squares) of rIFN-{gamma} (2.5 U/ml). CD1d (•, left) and class II MHC (•, right) expression were determined on day 3. Open symbols denote isotype control Ab staining. C, CD1d induction by rIFN-{gamma} and the Pam3Cys is TNF dependent. M{phi} were cultured for 24 h with rIFN-{gamma} (2.5 U/ml) and the TLR2 agonist Pam3Cys (1 ng/ml) in the presence of a blocking anti-TNF mAb (•) or an isotype-matched control mAb ({circ}). After 24 h, CD1d (left, p < 0.0001) and class II MHC (right, p = 0.0463) expression by F4/80+ M{phi} was analyzed by flow cytometry. Each symbol represents a separate experiment (line represents mean percentage of inhibition). D, CD1d up-regulation by rIFN-{gamma} and Mtb is TNF dependent. CD11b+ M{phi} were infected with Mtb (MOI 5:1) in the presence of rIFN-{gamma} (2.5 U/ml) and blocking anti-TNF mAb (•) or an isotype-matched control mAb ({circ}). After 24 h, M{phi} expression of CD1d (left, p = 0.0003) and class II MHC (right, p = 0.4427) was analyzed. Each symbol represents a separate experiment (line represents the mean percentage of inhibition).

 
When M{phi} infected at a low MOI were treated with rIFN-{gamma}, CD1d was induced on all cells, even though not all cells were infected (Fig. 1A). This result suggested that optimum CD1d induction requires a second soluble factor. We considered whether TNF modulates CD1d expression by M{phi} because of the consistent correlation between TNF production and CD1d induction and the IFN-{gamma}-dependence of TNF production (Fig. 3A). Although rTNF was not sufficient to up-regulate CD1d or MHC class II cell surface expression (Fig. 1C), we hypothesized that TNF and IFN-{gamma} may act synergistically to regulate CD1d expression. In the presence of rIFN-{gamma}, rTNF induced CD1d in a dose-dependent manner and at 10 ng/ml rTNF there was a 6.4-fold increase in surface levels (Fig. 3B). Although at the highest concentration of rTNF there was a 40% reduction in IFN-{gamma}-dependent class II MHC expression, M{phi} treated in this way still expressed high class II MHC levels compared with cells cultured with IFN-{gamma} and TLR agonists (compare Figs. 2B and 3B). Thus, in the presence of rIFN-{gamma}, neither rTNF nor viable Mtb dramatically alter class II MHC expression compared with uninfected M{phi} cultured with rIFN-{gamma} alone (Figs. 1A and 3B).

Purified M{phi} infected with Mtb, treated with heat killed bacteria or cultured with TLR ligands, up-regulates CD1d in the presence of rIFN-{gamma}. Under the same conditions, these various stimuli also induce TNF production. Although neither molecule induced CD1d alone, both TLR agonists and rTNF could synergize with IFN-{gamma} to induce CD1d. To directly test the role of TNF in CD1d regulation by TLR agonists or Mtb in combination with rIFN-{gamma}, the effect of TNF blockade on CD1d and class II MHC expression was examined. Blocking of TNF inhibited CD1d induction by Pam3Cys and rIFN-{gamma} (Fig. 3C) and by Mtb infection and rIFN-{gamma} (Fig. 3D) by ~65%, but had only a minor influence on MHC class II expression by M{phi} cultured in the absence of blocking anti-TNF Abs or isotype control Abs. We speculate that the residual induction of CD1d was mediated by rIFN-{gamma} alone and was resistant to inhibition by anti-TNF mAb, or that the blocking conditions were not 100% efficient. These experiments demonstrate that the physiological amounts of TNF produced by M{phi} following intracellular infection or after encounter with TLR ligands is sufficient to induce CD1d in the presence of rIFN-{gamma}. We conclude that signaling via IFN-{gamma}R and TLR is not sufficient to induce high CD1d levels on M{phi}, but also requires TNF production to mediate the effect. Taken together, these findings identify a mechanism that modulates CD1d expression during inflammation caused by microbial pathogens or other etiologies. Finding that the proinflammatory cytokine TNF can replace the requirement for microbial products in the induction of high CD1d levels on M{phi} may explain how noninfectious inflammatory conditions can regulate CD1d surface expression and promote the activation of self-reactive CD1d-restricted NKT cells.

CD1d is induced on M{phi} recruited to sites of inflammation in vivo

Our data show that in the presence of proinflammatory cytokines or microbial ligands, CD1d is up-regulated on inflammatory M{phi} in vitro. Given the important role of NKT cells in pulmonary immunity (28, 29, 30, 31), we designed an adoptive transfer experiment to measure CD1d induction on M{phi} recruited to sites of pulmonary inflammation. Purified M{phi} labeled with CFSE were adoptively transferred into either uninfected or Mtb-infected B6 recipients. The lungs were removed 48 h after cell transfer and donor CFSE+F4/80+ cells were detected by flow cytometry (Fig. 4A, left column). Higher levels of CD1d and class II MHC were induced on wild-type M{phi} transferred into infected recipients compared with the same cells transferred into uninfected recipient mice (Fig. 4A, top and middle row, and B). In parallel, to determine whether CD1d induction was dependent on IFN-{gamma} in vivo, highly purified F4/80+CD11b+ M{phi} obtained from IFN-{gamma}R–/– mice were transferred into infected B6 mice (Fig. 4, A, bottom row, and B). Whereas both CD1d and class II MHC were up-regulated on wild-type M{phi} recruited to infected lung tissue, minimal CD1d and class II MHC expression was observed on donor IFN-{gamma}R–/– M{phi} found in the lungs of infected recipient mice. These results confirm our in vitro data and show that CD1d is induced in an IFN-{gamma}-dependent manner on M{phi} at sites of inflammation in vivo.



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  		FIGURE 4. CD1d is up-regulated on M{phi} at the site of inflammation in vivo. A, Purified CFSE-labeled wild-type (WT) or IFN-{gamma}R–/– M{phi} were adoptively transferred into uninfected (top row) or Mtb-infected (middle and bottom row) mice. Transferred M{phi} identified in recipient lung tissue 48 h after cell transfer (CFSE+F4/80+) were analyzed for CD1d and class II MHC expression (thick lines). The thin lines represent control Ab staining. B, CD1d (left) and class II MHC (right) expression on transferred wild-type (WT) M{phi} from infected or uninfected recipients, or transferred IFN-{gamma}R–/– M{phi} from infected recipients using three or five mice per group. Bars show mean values ± SD. **, p < 0.001; ***, p < 0.001 by one-way ANOVA with Bonferroni post test. The difference in CD1d and class II MHC expression by wild-type M{phi} from uninfected recipients and IFN-{gamma}R–/– M{phi} from infected recipients was not statistically significant.

 
Level of CD1d cell surface expression on APC regulates NKT cell activity

Our results show that inflammatory signals regulate CD1d expression by M{phi} in vitro and in vivo. To determine whether M{phi} expressing higher levels of CD1d activate NKT cells more efficiently we tested a panel of CD1d-restricted NKT cell hybridomas. One group of iNKT cell hybridomas includes 24.7, 24.8, 24.9, and DN32.D3, which are CD1d-autoreactive (23, 24). All recognize {alpha}-GalCer presented by CD1d except 24.8, which instead recognizes cellular phospholipids (18, 20). A second group consists of CD1d-autoreactive hybridomas expressing a diverse TCR repertoire. Neither 14S.6 nor 14S.15 express an invariant TCR or recognize {alpha}-GalCer; instead both hybridomas recognize CD1d-transfected tumor cell lines including RMA-S, but the CD1d-restricted Ags remain unidentified (18, 23). The third group includes {alpha}-GalCer-reactive iNKT cell hybridomas KT/7, KT/12, KT/22, and KT/23, which are not known to be autoreactive but show strong reactivity to {alpha}-GalCer presented by CD1d (18).

M{phi} were cultured in medium alone, or treated with rIFN-{gamma}, Pam3Cys, or both before the hybridomas were added. Because the combination of rIFN-{gamma} and Pam3Cys is the most potent inducer of CD1d we expected these M{phi} to present CD1-restricted Ags more efficiently to the NKT cell hybridomas (Fig. 5A). For the {alpha}-GalCer-specific CD1d-restricted NKT cell hybridomas, Gal({alpha}1->2)GalCer ({alpha}-GalGalCer) was used as a model exogenous Ag (Fig. 5B and data not shown) (12). This ceramide requires uptake and lysosomal processing by {alpha}-galactosidase A to convert it into {alpha}-GalCer (32). For the CD1d-autoreactive NKT cell hybridomas, the M{phi} were plated at different cell densities in the absence of Ag (Fig. 5C and data not shown). Interestingly, all three groups of NKT cell hybridomas significantly recognized only the M{phi} treated with a combination of rIFN-{gamma} and Pam3Cys.



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  		FIGURE 5. CD1d cell surface expression regulates NKT cell activation. A, Four groups of M{phi} were compared for their ability to stimulate CD1d-restricted NKT cell hybridomas. Purified M{phi} were cultured in medium alone, with rIFN-{gamma} (2.5 U/ml), Pam3Cys (1 ng/ml), or both for 3 days. The dot plots show representative F4/80 and CD1d expression by M{phi} used as APC in the T cell hybridoma assays. B, CD1d presentation of an exogenous Ag was tested using {alpha}-GalGalCer. M{phi} were pulsed with {alpha}-GalGalCer at the indicated concentrations before culture with {alpha}-GalCer-reactive NKT hybridomas. Treatment conditions for the M{phi} medium alone ({circ}), Pam3Cys ({blacktriangledown}), rIFN-{gamma} ({blacksquare}), and rIFN-{gamma}+Pam3Cys (•). C, M{phi} treated as in A and B were tested for their recognition by CD1d-autoreactive hybridomas. D–F, RMA-S sublines that differed in their cell surface CD1d levels were used as APC for {alpha}-GalCer-reactive (D and F) or autoreactive (E) CD1d-restricted NKT cell hybridomas. IL-2 production (picogram per milliliter) by the NKT cell hybridomas was measured by ELISA after 24 h. One representative experiment of two to six is shown.

 
The results obtained using M{phi} as APC support our model that CD1d cell surface expression can regulate NKT cell activity. Although the activation of T cell hybridomas is not dependent upon accessory signals, treatment of M{phi} with IFN-{gamma} and Pam3Cys is likely to induce changes that may affect their function in ways that are independent of CD1d surface levels. To conclusively show that CD1d cell surface expression affects NKT cell activation, we sorted CD1d-transfected RMA-S cells (RMA-S.CD1d) to obtain sublines that stably express different CD1d levels and used these sublines as APC for the NKT cell hybridoma assays. A direct correlation was observed between the CD1d level expressed by the RMA-S cells and the amount of IL-2 produced by three CD1d-autoreactive and three {alpha}-GalCer-reactive NKT cell hybridomas (Fig. 5, D–F). Importantly, a clear dose response was observed for the activation of NKT cell hybridomas by RMA-S.CD1d sublines expressing only moderate CD1d levels (MFI = 25–100) (Fig. 5F). These lower levels of CD1d were comparable to the physiological levels of CD1d expressed by the M{phi} used in our experiments. Thus, in agreement with our model, APC expressing higher levels of cell surface CD1d activate NKT cell hybridomas more efficiently.

In conclusion, it appears that CD1d levels contribute to the activation of both invariant and TCR diverse NKT cells. Furthermore, this was true not only for autoreactive CD1d-restricted NKT cell hybridomas, but also for {alpha}-GalCer-specific NKT cell hybridomas. These data suggest that even in the presence of a potent Ag, the CD1d-TCR avidity must surpass a minimum threshold to activate the NKT cell. Such a threshold makes possible the regulation of NKT cell activation by modulating the expression of CD1d on APC.

CD1d levels affect activation of primary NKT cells in vitro and in vivo

To determine whether CD1d levels affect the activation of primary NKT cells, enriched thymic NKT cells were tested for their ability to recognize {alpha}-GalCer-pulsed target cells. Thymic NKT cells recognized rIFN-{gamma} plus Pam3Cys-treated M{phi} expressing high CD1d levels, better than M{phi} cultured in medium alone (Fig. 6A). Thus, a higher CD1d level expressed by M{phi} enhances Ag recognition and activation of primary NKT cells. The capacity of thymic NKT to recognize RMA-S cells expressing different CD1d levels was also tested. Similar to our findings using NKT cells hybridomas, thymic NKT cell activation, as measured by their production of IL-4 and IFN-{gamma}, correlated with the CD1d level expressed by the RMA-S cells (Fig. 6B and data not shown). It is noteworthy that IL-4 and IFN-{gamma} production by primary iNKT cells in vitro required exogenous IL-2. IL-2 has also been shown to affect the generation of cytokine producing iNKT cells in vivo (25). The requirement for exogenous IL-2 in vitro may reflect that the APC used in the culture system fail to produce IL-2 or fail to provide a costimulatory signal that leads to IL-2 production by the iNKT cells. Finally, we asked whether NKT cell activation in vivo was affected by the CD1d level expressed by APC. To answer this question, splenic iNKT cells specific for {alpha}-GalCer were enumerated by an IFN-{gamma} ELISPOT assay following i.v. administration of {alpha}-GalCer-pulsed M{phi} that had been cultured in medium alone or in the presence of rIFN-{gamma} and Pam3Cys (25). Injection of M{phi} expressing higher CD1d levels elicited a greater expansion of iNKT cells compared with M{phi} expressing lower CD1d levels (Fig. 6C). For example, whereas 43 spot forming cells/105 cells were detected after injection of {alpha}-GalCer-pulsed CD1dlow M{phi}, this number increased 2.5-fold to 109 spot forming cells/105 cells after injection of {alpha}-GalCer-pulsed CD1dhigh M{phi}. Taken together, these data show that the cell surface level of CD1d expressed by M{phi} influences the activation of primary iNKT cells in vitro and in vivo.



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  		FIGURE 6. CD1d levels affect activation of primary NKT cells in vitro and in vivo. A, Two groups of M{phi} were compared for their ability to stimulate enriched thymic NKT cells. Purified M{phi} were cultured in medium alone (untreated) or with rIFN-{gamma} (2.5 U/ml) and Pam3Cys (P3C) (1 ng/ml) for 3 days. M{phi} were pulsed with {alpha}-GalCer (100 ng/ml) or vehicle before culture with enriched thymic NKT cells in the presence of rIL-2. IL-4 (mean ± SD) was measured in the culture supernatants after 48 h by ELISA. **, p < 0.01; ***, p < 0.001. B, RMA-S sublines that differed in their cell surface CD1d levels were pulsed with {alpha}-GalCer (10 ng/ml, closed symbols) or vehicle (open symbols) and used as APC with enriched thymic NKT cells in the presence (squares) or absence (circles) of rIL-2. IL-4 (mean ± SD) was measured in the culture supernatants after 48 h by ELISA. C, M{phi} treated as in A were tested for their capacity to stimulate expansions of activated NKT cells in vivo. {alpha}-GalCer-pulsed M{phi} were injected i.v. into mice and after 48 h the spleen was retrieved. Splenocytes were treated with {alpha}-GalCer (+) or vehicle (–) and the number of IFN-{gamma}-secreting cells (spot forming cells (SFC)/105) was enumerated by an ELISPOT assay. ***, p < 0.001. ns, Not significant. These results are representative of two to three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Both CD1d and class II MHC traffic through endosomal pathways and present exogenous Ags to T cells. However, our comparison of CD1d and class II MHC expression by inflammatory M{phi} reveals a fundamental difference in their regulation. Maximal cell surface expression of class II MHC on M{phi} is achieved using IFN-{gamma} alone; addition of TLR agonists inhibits maximal class II MHC induction by IFN-{gamma}. In contrast, maximal CD1d expression requires two signals: one provided by IFN-{gamma} and the second provided by either TNF or TLR agonists. In vitro infection of M{phi} with live bacteria in the presence of exogenously added IFN-{gamma} led to dramatic up-regulation of CD1d. CD1d levels are also modulated in vivo as we observe that CD1d is up-regulated on M{phi} recruited to the lungs of Mtb-infected mice. On average, the CD1d induction we observed on M{phi} transferred into Mtb-infected recipient mice was lower than what we observed on M{phi} in vitro after treatment with rIFN-{gamma} and bacterial products or TNF. Transferred M{phi} may not encounter high enough concentrations of bacteria or TNF in the lung tissue to induce high levels of cell surface CD1d. Although we observed that live bacteria had little effect on IFN-{gamma}-induced class II MHC expression, our finding that dead bacteria or TLR agonists inhibit IFN-{gamma}-induced class II MHC up-regulation are in agreement with the literature (27, 33). These results emphasize how differently CD1d is regulated because it is induced by the same conditions that inhibit class II MHC expression. Down-regulation of IFN-{gamma}-induced class II MHC by TLR ligands may be one mechanism by which microbes evade class II MHC-restricted immune responses. The inhibition observed using pure TLR ligands may be due to high concentrations in the in vitro cultures that do not accurately reflect the amount of TLR ligands exposed to the M{phi} when infected with live bacteria. Alternatively, other microbial products expressed by viable bacteria may counter the inhibitory effects of TLR agonists. In contrast, TLR agonists are potent inducers of CD1d in combination with IFN-{gamma} at physiological concentrations. Maximum CD1d induction was observed using 1 ng/ml LPS or Pam3Cys, the lowest concentration tested. Induction of CD1d by TLR agonists may therefore be achieved in the picogram per milliliter range where the effect on class II MHC expression is less pronounced. Interestingly, we found the effect of TLR ligands on CD1d induction is mediated by TNF. The ability of IFN-{gamma} and TNF to act synergistically in CD1d up-regulation is important as it suggests that even in the absence of infection, inflammation can induce CD1d, which in turn can promote activation of CD1d-autoreactive NKT cells.

CD1d-restricted NKT cells are believed to be an early source of cytokines such as IL-4 and IFN-{gamma} in various disease models. Whether IFN-{gamma} is required for optimum CD1d induction and subsequent NKT cell activation, this paradigm suggests that there must be another cellular source of early IFN-{gamma} production. Neutrophils and M{phi} appear to be the primary source of IFN-{gamma} following Salmonella infection, and they outnumber IFN-{gamma}-producing NK, NKT, and T cells (34). Furthermore, Brigl et al. (21) show that in addition to iNKT cells, the main IFN-{gamma}-producing lymphocyte population during the first 3 days of Salmonella infection is CD3– and potentially NK cells.

Determining the cellular requirements that lead to NKT cell activation is essential for understanding how NKT cells affect immunity to tumors, infection, and to self. We find that CD1d up-regulation promotes activation of NKT cells both in vitro and in vivo. We find this to be true for both invariant and diverse CD1d-restricted NKT cell hybridomas, including ones that are autoreactive and ones that recognize exogenous Ag, indicating that this mechanism is independent of the Ag presented by CD1d. We envision that increasing CD1d levels favor NKT cell activation by increasing the avidity of the CD1d-NKT cell interaction. The avidity of the TCR-CD1d interaction is known to be important for NKT cell activation. The reduced potency of {alpha}-GalCer analogues to activate iNKT cells correlates with reduced avidity of the invariant TCR for the Ag-CD1d complex (35). In addition to the Ag, the TCR repertoire also influences the avidity of the CD1d-TCR interaction (36, 37). An increase in the CD1d-TCR avidity may explain why iNKT cells expressing V{beta}8 are disproportionately deleted during T cell ontogeny in mice overexpressing CD1d (38). CD1d levels also affect tissue inflammation in vivo. CD1d-restricted NKT cells have the capacity to prevent or ameliorate some forms of experimental autoimmunity. In the NOD mouse model of human type I diabetes, NKT cells are dysfunctional and correction of this defect by adoptive transfer of NKT cells, {alpha}-GalCer administration, or overexpression of a CD1d-restricted TCR protects NOD mice from diabetes (39, 40). Recently, overexpression of CD1d specifically within the pancreatic islets of NOD mice was found to rescue them from diabetes (41). By increasing the number of CD1d-self-Ag complexes on the cell surface, the avidity for the TCR may be sufficiently raised to trigger NKT cell activation under physiological conditions. These experiments support our hypothesis that the cell surface CD1d level modulates NKT cell activation in vivo.

The advent of mouse models that allow the transient ablation of DC in adult animals has allowed investigators to determine the in vivo requirement for DC during immune responses (42, 43, 44). These models have been used to show that splenic NKT cell activation is dependent upon DC following systemic administration of {alpha}-GalCer in vivo (45, 46). Interestingly, in the liver, Kuppfer cells, and not DC, are the critical APC for iNKT cell activation (45). Thus, whether DC or M{phi} are required for NKT cell activation may depend on the tissue, the resident cell types, and the degree of cellular activation. For example, although diphtheria toxin-mediated DC deletion leaves the splenic M{phi} population untouched, splenic M{phi} do not support rapid iNKT cell activation, and even ex vivo, splenic M{phi} were not efficient APC for iNKT cells (46). Although these results may at first appear to contradict our data, they are in fact consistent because we find that highly purified unstimulated M{phi} poorly stimulate NKT cell hybridomas and primary NKT cells. However, we clearly show that the inflammatory cytokines IFN-{gamma} and TNF lead to CD1d up-regulation on M{phi}, which makes them effective at activating NKT cells both in vitro and in vivo.

In addition to inducing CD1d, treatment of M{phi} with rIFN-{gamma} and bacterial products or TNF is likely to up-regulate the expression of costimulatory molecules such as CD40, CD80, and CD86, and soluble mediators such as IL-12, which may promote NKT cell activation. Our data supports the conclusion that the CD1d surface level modulates NKT cell activation. First, the CD1d levels correlated with activation of CD1d-restricted T cell hybridomas irrespective of their Ag specificity or TCR type. Importantly, the activation of these CD1d-restricted T cell hybridomas is independent of soluble factors and cell-cell contact dependent accessory signals (18). Furthermore, we derived sublines of CD1d-transfected RMA-S tumor cells that express different cell surface levels of CD1d. This allowed us to directly correlate the cell surface CD1d level with NKT cell activation, independently of any potential differences in expression of costimulatory signals that may affect NKT cell activation. This allowed us to circumvent the need to use rIFN-{gamma} and bacterial products to induce CD1d, and enabled us to isolate the contribution of CD1d levels to NKT cell activation.

The capacity of self-lipid Ags to activate CD1d-restricted NKT cells may be dependent on certain accessory signals. The activation of iNKT cells by Salmonella is an instructive example. Our current understanding is that Salmonella LPS activates DC by MyD88-dependent TLR signaling resulting in IL-12 secretion (16, 21). IL-12 costimulates iNKT cell recognition of the CD1d-restricted endogenous self-lipid Ag iGb3 (16). CD1d induction could play an important role in augmenting NKT cell activation by weak endogenous Ags. This result is true for the 24.8 NKT cell hybridoma that recognizes phospholipids, as well as for other autoreactive NKT cell hybridomas that possibly recognize iGb3. Importantly, iNKT cell activation and IFN-{gamma} secretion stimulated by Salmonella LPS is predicted to increase cell surface CD1d expression and lead to a positive feedback loop that would further stimulate iNKT cell activation.

For microbial Ags that are presented by CD1d, the affinity of the iNKT TCR for the CD1d-Ag complex may be sufficiently high that additional accessory signals are unnecessary. NKT cells recognition of DC presenting CD1d-restricted Sphingomonas Ags is independent of MyD88, and presentation of Leishmania Ags is independent of IL-12 (14, 16). Thus, additional costimulation may not be required for the activation of iNKT cells by bona fide CD1d-restricted foreign lipid Ags. Nevertheless, CD1d induction may enhance Ag recognition because our experiments show that greater iNKT cell activation can be achieved even for very potent Ags such as {alpha}-GalCer.

We favor a model in which lipid Ags promote formation of stable CD1d-Ag-TCR ternary complexes. Up-regulation of CD1d cell surface levels would enhance the initial APC-NKT cell interaction and promote full NKT cell activation. The Ags may be of microbial origin, or self-lipids that are normally sequestered or become altered during cellular stress. In the present study examining NKT cell activation following infection, we found that proinflammatory mediators regulate cell surface CD1d expression on M{phi}. Because these mediators could be of either microbial or cellular origin, we hypothesize that regulation of CD1d cell surface expression is a general mechanism by which the immune system controls NKT cell activation and function. APC expressing higher CD1d levels activate CD1d-restricted NKT cells more efficiently. Once a stable interaction between APC and NKT cell is established, we envision that contact dependent costimulatory signals and soluble factors such as CD40/CD40L and IL-12 may be required to fully activate CD1d-restricted NKT cells. Our model provides important insight into how CD1d is regulated and how CD1d levels expressed by APC influence NKT cell activation following infection and possibly during other inflammatory conditions.


   Acknowledgments
 
We acknowledge helpful scientific discussions with Michael Brenner and thank Mi Xiao Donovan for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R03 AI49093 and R01 HL80312 and by an Arthritis Foundation Investigator Award (to S.M.B.). G.S.B. is a Lister-Jenner Research Fellow and acknowledges support from The Medical Research Council Grants G9901077 and G0400421 and The Wellcome Trust Grant 072021/Z/03/Z. Back

2 Address correspondence and reprint requests to Dr. Samuel M. Behar, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Smith Building Room 516B, One Jimmy Fund Way, Boston, MA 02115. E-mail address: sbehar{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; M{phi}, macrophage; {alpha}-GalCer, {alpha}-galactosylceramide; iNKT, invariant NKT; MFI, mean fluorescence intensity; MOI, multiplicity of infection. Back

Received for publication May 17, 2005. Accepted for publication June 28, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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